Role of nitric oxide and prostaglandins in the potentiating effects of calcitonin gene-related peptide on lipopolysaccharide-induced interleukin-6 release from mouse peritoneal macrophages

Previous data from our laboratory have shown that calcitonin gene-related peptide (CGRP) has a potentiating effect on lipopolysaccharide-(LPS) induced interleukin-6 (IL-6) release from mouse macrophages. However, the mechanism of this effect was not clear. Since the nitric oxide (NO) and prostaglandins (PGs) induced by LPS might modulate IL-6 release, we examined whether NO and PGs were also involved in the potentiating effect of rat CGRP (rCGRP) on LPS-induced IL-6 release from mouse macrophages. The IL-6 level in the medium was measured by enzyme-linked immunosorbent assay. Accumulation of NO was assessed by measuring the presence of nitrite by the Greiss reaction. PGI2 was assessed by measuring the formation of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) by radioimmunoassay. The results showed that the potentiating effect of rCGRP (0.1 nm) on LPS-induced IL-6 release was significantly inhibited by either 100 micrometers NG-monomethyl-L-arginine acetate (L-NMMA; an inhibitor of NO synthase) or 10 micrometers indomethacin (an inhibitor of cyclo-oxygenase). The LPS-induced NO and PGI2 production from these cells was increased significantly by rCGRP at 0.01-10 nm in a concentration-dependent manner, which was blocked by L-NMMA and indomethacin. These results suggest that rCGRP enhances the NO production elicited by LPS and subsequently increases the PGs production which is involved in the potentiating effect of rCGRP on LPS-induced IL-6 release from the peritoneal macrophages in the mouse.     

Central cardiovascular effects of calcitonin gene-related peptide: interaction with noradrenaline in the nucleus tractus solitarius of rats

Summary The haemodynamic responses to microinjections of rat or human calcitonin gene-related peptide (CGRP) into the nucleus tractus solitarius (NTS) of rats were studied. 40 fmol rCGRP did not significantly modify cardiovascular parameters, but 0.2 pmol decreased blood pressure and heart rate (HR), whereas 2 pmol produced a pressor response with no effect on HR. hCGRP elicited a transient fall in blood pressure when administered at the highest dose (2 pmol), but had no effects when given at 0.2 pmol. A possible functional relationship with catecholamines was also investigated. The hypotensive response to 20 nmol noradrenaline (NA) was significantly modified by simultaneous administration of a low dose (40 fmol, ineffective alone) of rCGRP. When rCGRP (40 fmol) was coinjected simultaneously with an ineffective dose (10 pmol) of NA, a hypotensive response was observed. Our results provide evidence that rCGRP may play a role in the control of cardiovascular homeostasis in the NTS, and suggest a functional interaction between this peptide and NA.     

The short term effect of peripherally administered brain-gut peptides on gastric acid secretion in rats

Certain brain gut-peptides are known to either stimulate or inhibit gastric acid secretion in several species after direct injection into the central nervous system. However there is inconsistency of published results on the gastric acid secretory response to some of these peptides after peripheral administration in different experimental systems. Seven peptides, namely neurotensin (NT), substance P, cholecystokinin (CCK), thyrotropin releasing hormone (TRH), human calcitonin (hCT), rat calcitonin-gene-related peptide (rCGRP) and bombesin, all known to modulate gastric acid secretion after central administration, were initially screened for activity after peripheral (subcutaneous) injection of 10 g/kg body weight in a single rat model. Peptides showing an effect were retested at lower doses. Despite the inherent variability of the gastric acid secretory response in the non-anaesthetized pylorus ligated rat, a standardized experimental design confirmed that reproducible and statistically valid results could be obtained. The technical feasibility of using a one hour collection period as might be appropriate for short acting peptides was demonstrated by the significant dose dependent inhibitory activity of salmon calcitonin. In this model, NT and substance P had no significant effect on either volume or concentration of acid secreted, CCK showed a slight stimulation of acid output, and TRH, hCT, rCGRP and bombesin all inhibited acid output; CGRP and bombesin were active at 10 and 100-fold lower doses. The potent and inhibitory activity of bombesin in this system is in disagreement with other publications reporting no effect or variable stimulatory effect in rats. Time and dose dependent responses in our rat system indicate that this apparent discrepancy may be explained by the short duration of action of bombesin.     

Effect of lanthanum ions on the amplitude distributions of miniature endplate potentials and on synaptic vesicles in frog neuromuscular junctions

Miniature endplate potential (MEPP) amplitude distributions, MEPP frequencies and percentages of small MEPPs were determined as well as synaptic vesicle diameters and numbers in the frog neuromuscular junction during La3+ treatment. MEPP frequencies initially increased by two orders of magnitude and then fell to very low values. La3+ treatment had an initial postsynaptic effect making the MEPPs larger. Prolonged treatment had a variable effect on MEPP amplitudes. There were considerable variations in MEPP frequencies in adjacent junctions so single junctions on edge muscle fibers were recorded for the duration of many experiments and later identified in the electron microscope. Therefore, the physiological and morphological conditions of a given identified junction could be compared. There was a loss of synaptic vesicles and no change in mean diameter during depletion. During high MEPP frequencies infoldings occurred on the axolemma and these disappeared when MEPP frequencies decreased towards the end of the La3+ treatment. After 3-4 h of La3+ treatment, the overall frequency of MEPPs dropped and many were composed of a small class of MEPPs. It is suggested that the morphological correlate of small MEPPs, as well as the classical bell-MEPPs is likely to be synaptic vesicles.     

A new type of transmitter release at the neuromuscular junction

Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs. In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs. The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude. It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes. The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs. In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter. In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind. It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.     

Effects of hexamethonium on transmitter release from the rat phrenic nerve.

Hexamethonium (HEX) was applied to isolated transected diaphragm-phrenic nerve preparations of the rat in order to further elucidate the functional role of the presynaptic nicotinic autoreceptors. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the neuromuscular junctions in the presence and absence of HEX to determine the relative effect of this nicotinic antagonist on end-plate sensitivity and evoked release. In this study we show that HEX enhances transmitter release for the first few stimuli, but this action is not maintained during a train-of-six stimulation. While these results support the hypothesis that transmitter released from the nerve terminal normally has a negative feedback effect by depressing transmitter release it is proposed that HEX also has secondary actions on the neuromuscular junction that are unrelated to autoreceptor blockage. The results with HEX suggests that the presynaptic receptors may differ pharmacologically from the postsynaptic receptors.     

Effects of cesium ions on the frequency of miniature end-plate potentials at the frog neuromuscular junction

The effects of Cs ions on the frequency of miniature end-plate potentials (MEPPs) were investigated at the frog neuromuscular junction. The frequency of MEPPs increased gradually with time, after a delay, when part of the NaCl in normal Ringer's solution was replaced isotonically with CsCl. The delay was greatly prolonged and the rate of increase in MEPP frequency slowed when a low concentration--between 6 and 40 mM--of CsCl was applied. As the external concentration of CaCl2 (0.4-6.3 mM) was increased, the delay was prolonged and the rate of increase became slower. The effect of Mg ions was qualitatively the same as that of Ca ions. In the case of reapplication, Cs ions produced a prompt increase in MEPP frequency and the rate was much faster than before. When the solution contained no added Ca and 1 mM EGTA, no appreciable change in MEPP frequency was observed when CsCl was applied. It is suggested that the first step in the gradual increase in MEPP frequency is the slow entry of Cs ions into the nerve terminal, following which transmitter release is mainly induced by the influx of external Ca ions.     

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.     

Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.

A study was made to determine the disposition of vesicle-associated proteins (syntaxin, SV2, SNAP-25) and calcium channels with respect to the spatial extent of spontaneous and evoked quantal release within regions of amphibian motor-nerve terminal branches delineated by FM1-43 stained vesicle clusters (blobs). Discrete concentrations of vesicles revealed approximately 2 microm apart along the length of terminal branches through FM1-43 staining were identical in size and spacing to those identified along terminal branches with SV2 antibody (AbSV2). Fluorescent antibodies to syntaxin 1 (AbS), SNAP-25 (AbS25) and the calcium channel alpha1B subunit (Abalpha1B) were found in relatively high concentrations coincident with the AbSV2 blobs. Three extracellular recording electrodes were placed in the vicinity of individual FM1-43 blobs, and an algorithm was used to determine the spatial origin of miniature endplate potentials (MEPPs) and EPPs together with their relative amplitudes. MEPPs and EPPs originated throughout the region stained by FM1-43 but not elsewhere; amplitude-frequency distributions of MEPPs and EPPs were similar for all FM1-43 blobs with average coefficients of variation of no less than 0.28. A linear relationship existed between the size of an FM1-43 blob, measured as the integrated extent of FM1-43 staining of a blob, and the frequency of MEPPs as well as the probability of EPPs from the blob. There was a proximo-distal gradient in the size of FM1-43 blobs along the length of single terminal branches, suggesting a gradient in release probability along the branches. The frequency distribution of the distances between blobs was approximately Gaussian, whereas the frequency distribution of the size of blobs was highly skewed and was best fitted with a gamma distribution. It is concluded that there are correlations among the extent of labeling of SNAP-25, syntaxin and calcium channels at a release site, the store of vesicles to be found there, and the probability of spontaneous and evoked quantal release.     

Effects of bis(7)-tacrine on spontaneous synaptic activity and on the nicotinic ACh receptor of Torpedo electric organ.

Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two tacrine molecules are linked by a heptylene chain. We tested the effects of bis(7)-tacrine on the spontaneous synaptic activity. Miniature endplate potentials (MEPPs) were recorded extracellularly on slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and half-width. We also tested the effect of bis(7)-tacrine on nicotinic acetylcholine receptors by analyzing the currents elicited by acetylcholine (100 microM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine inhibited the acetylcholine-induced currents in a reversible manner (IC(50) = 162 nM). The inhibition of nicotinic acetylcholine receptors was not voltage dependent, and bis(7)-tacrine increased the desensitization of nicotinic acetylcholine receptors. The Hill coefficient for bis(7)-tacrine was -0.72 +/- 0.02, indicating that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1, but does not affect the binding of alpha-bungarotoxin with the nicotinic acetylcholine receptor. In conclusion, bis(7)-tacrine greatly increases the spontaneous quantal release from peripheral cholinergic terminals at a much lower concentration than tacrine. Bis(7)-tacrine also blocks acetylcholine-induced currents of Torpedo electric organ, although the mechanism is different from that of tacrine: bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.     

Effects of two synaptic activators, calcium and ethanol, on MEPP distribution in time.

Miniature end-plate potentials (MEPPs) were recorded intracellularly from sartorius muscle of Rana esculenta. Tracings were divided into time bins whose duration approximated one-fifth of the mean interval between consecutive potentials. The observed number of bins containing 0, 1, 2, ... MEPPs was compared, by the X2 test, with the number calculated from the Poisson equation. MEPP timing was analyzed in the absence as well in the presence of Ca2+ (1 mM, 2.5 MM, and 15 mM). In half of the experiments, 0.5% ethanol was added to the bathing solution. In the absence of Ca2+, MEPP timing fitted the Poisson predictions. On adding Ca2+, the fit became poor and MEPPs showed the tendency to cluster. At 15 mM Ca2+, no experiment proved to be Poissonian. Though increasing the frequency of MEPPs similarly to Ca2+, ethanol maintained a Poissonian release of transmitter at any concentration of Ca2+. It is suggested that ethanol masks the effects of Ca2+ on MEPP timing by also inducing the discharge of transmitter outside the Ca2+-dependent sites of exocytosis in the presynaptic membrane.     

Suppressive effect of genistein on rat bone osteoclasts: involvement of protein kinase inhibition and protein tyrosine phosphatase activation

The suppressive effect of genistein on osteoclast-like multinucleated cells from rat femoral tissues was investigated. The bone cells isolated from rat femoral tissues were cultured for 48 h in an alpha-minimal essential medium (5% fetal bovine serum) containing either vehicle or genistein (10(-7)-10(-5) M). Osteoclasts were estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of genistein caused a significant decrease in the number of osteoclasts. Such a decrease was also seen in the presence of calcium choride (10(-5) M). Magnesium chloride (10(-5)-10(-3) M), a blocker of Ca2+ channels, had no effect on the number of osteoclasts. The effect of genistein (10(-5) M) or calcium (10(-3) M) in decreasing osteoclasts was significantly prevented by the presence of magnesium (10-3 M). Vanadate (10(-6)-10(-4) M), an inhibitor of protein tyrosine phosphatase activity, did not have an effect on the number of osteoclasts. The genistein's effect was not altered by vanadate. When isolated osteoclasts were cultured for 24 h in the presence of genistein (10(-7)-10(-5) M), protein kinase activity in the 5500 g supernatant of homogenate of the cells was significantly decreased, while protein tyrosine phosphatase activity was significantly elevated. Such an effect was also seen by the addition of genistein (10(-7)-10(-5) in the enzyme reaction mixture in vitro. The present study suggests that the suppressive effect of genistein on rat bone osteoclasts is partly involved in the inhibition of protein kinase and the activation of protein tyrosine phosphatase in osteoclasts.     

The possible involvement of hyperpolarizing mechanisms in histamine-induced relaxation of the rat portal vein

The present study evaluated the effects of histamine 10(-2) M on longitudinal preparations of rat portal vein. It was observed that histamine 10(-2) M induced relaxation of rat portal vein preparations pre-contracted with phenylephrine 10(-4) M. On the other hand, no pharmacological effects were observed in preparations not pre-contracted. The observed histamine-induced relaxing effect was absent in preparations pre-contracted with KCl (120 mM) or in the presence of depolarizing nutritive solution. However, the histamine-induced relaxation was still present in the endothelium-removed preparations. The histamine-induced relaxation also was not prevented by astemizole (10(-6) M, 10(-5) M and 10(-4) M), cimetidine (10(-5) M, 10(-4) M and 10(-3) M) or thioperamide (10(-6) M, 10(-5) M and 10(-4) M), selective antagonists H(1), H(2) and H(3), respectively. The presence of L-NAME 10(-4) M or L-NAME 10(-4) M plus indomethacin 10(-5) M also did not prevent the histamine-induced relaxation observed in rat portal vein. Thus, the histamine-induced relaxation observed in rat portal vein appears to involve a non-endothelial hyperpolarizing mechanism independent of H(1), H(2) and H(3) receptors.     

On the possible origin of “giant or slow-rising” miniature end-plate potentials at the neuromuscular junction

Giant or slow-rising miniature end-plate potentials (GMEPPs) caused by vesicular release of acetylcholine (ACh) occur at any time in about 50% of mouse diaphragm neuro muscular junctions, but generally at frequencies less than 0.03 s^–1. Their frequency is, unlike that of miniature end-plate potentials (MEPPs), not affected by nerve terminal depolarization. Unlike MEPPs and stimulus-evoked end-plate potentials, GMEPPs have a prolonged time-to-peak and show an increase in time-to-peak with amplitude. By using these differences in amplitude and time course, GMEPPs can be separated from MEPPs. In contrast to MEPPs, GMEPPs are not blocked by botulinum neurotoxin type A. GMEPPs have a greater temperature sensitivity than MEPPs, disappearing at temperatures below 15°C. Long-term paralysis by botulinum toxin and certain drugs which inhibit protein kinase C or affect actin filament polymerization (cytochalasins) enhance the frequency of GMEPPs. End-plate current recordings show that similar postsynaptic ACh receptors are activated by MEPPs and GMEPPs. It is suggested that GMEPPs are not caused by mechanisms involved in regulated neurotransmitter release but are generated by constitutive secretion.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.

The effect of Ca2+-binding protein regucalcin on neutral phosphatase activity in rat brain cytosol was investigated. Phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The presence of calcium chloride (10(-5) and 10(-4) M) in the enzyme reaction mixture caused a significant increase in phosphatase activity toward three phosphoaminoacids. The enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (1 or 5 microg/ml) in the presence of calcium (10(-5) M). Such an effect was not seen in the presence of phosphotyrosine. Trifluoperazine (2x10(-5) M), an antagonist of calmodulin, completely inhibited calcium (10(-5) M)-increased phosphatase activity toward phosphoserine and phosphothreonine, whereas it had no effect on the enzyme activity toward phosphotyrosine. Regucalcin (10(-9) M) significantly inhibited phosphatase activity toward three phosphoaminoacids without or with Ca2+ addition. The inhibitory effect of regucalcin (10(-10) and 10(-9) M) was also seen in the presence of Ca2+ (10(-5) M) and calmodulin (5 microg/ml). The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of phosphatase activity toward three phosphoaminoacids; this effect was completely abolished by addition of regucalcin (10(-9) M). The present study suggests that the endogenous regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.     

Insulin-like growth factor 1 stimulates the release of acetylcholine from rat cortical slices

The effect of somatomedin, or insulin-like growth factors (IGF-1 and IGF-2), on the basal and potassium induced release of [3H]acetylcholine ([3H]Ach) from rat cortical slices, previously preincubated with [3H]choline ([3H]Ch), was studied in vitro. IGF-1 (1.4 x 10(-9) to 1.4 x 10(-8) M) had no effect on the basal release of [3H]ACh, while IGF-1 (1.4 x 10(-9) to 4.3 x 10(-8) M) increased the potassium induced release of [3H]ACh from rat brain slices in a concentration-dependent manner. However IGF-2 (1.4 x 10(-8) M) had no effect. Insulin (1.8 x 10(-8) to 5.3 x 10(-8) M), similarly, did not have any influence on the release of [3H]ACh, demonstrating that the facilitatory effect of IGF-1 on [3H]ACh release is not mediated via insulin receptors. This report demonstrates for the first time that IGF-1 has an effect on neurotransmission in the adult brain.     

Mechanism of action of an antiallergic agent, amlexanox (AA-673), in inhibiting histamine release from mast cells. Acceleration of cAMP generation and inhibition of phosphodiesterase

Amlexanox markedly inhibits histamine release from rat mast cells. To clarify the mechanism of this inhibition, we investigated the effect of amlexanox on cAMP content, which, when increased, inhibits histamine release in rat peritoneal mast cells. At concentrations of 10(-8)-10(-6)M, amlexanox or isoproterenol increased the cAMP content of mast cells over that of control cells about 2-fold. When the mast cells were incubated with 10(-8), 10(-7) and 10(-6) M of amlexanox combined with 10(-7) M isoproterenol, the cAMP contents were synergistically increased 15-, 60- and 88-fold, respectively. 3-Isobutyl-1-methylxanthine (IBMX) at 10(-6)-10(-4) M increased the cAMP content 1.7-3.8-fold, and a combination of 10(-4) M IBMX and 10(-7) M isoproterenol synergistically increased the cAMP content 41-fold. A combination of amlexanox and IBMX synergistically increased the cAMP content 19-fold. The increase in cAMP content, when amlexanox and isoproterenol were combined, was transient; it peaked at 0.5 min after the drugs were administered, then decreased to 20-30% of the peak value about 2 min later. Pretreatment of mast cells with amlexanox reduced the effect of the combination of amlexanox and isoproterenol, indicating tachyphylaxis; pretreatment with IBMX had no such effect. The cAMP content of macrophages was also increased by amlexanox, but when combined with isoproterenol or PGE2, the effect was additive. Amlexanox inhibited cAMP phosphodiesterase in rat mast cells; its IC50 value was 1.4 X 10(-5) M, and its inhibitory activity was half that of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of protein kinase activity in the cytosol of regenerating rat liver: regulatory role of endogenous regucalcin

The effect of regucalcin, a Ca2+-binding protein, on protein kinase activity in the cytosol of regenerating rat liver was investigated. Protein phosphorylation was significantly increased in the liver cytosol obtained at 6, 24, and 48 h after a partial hepatectomy (about 65%) in comparison with that of sham-operated rats. This increase was significantly inhibited by the addition of trifluoperazine (2x10(-5) M), staurosporine (10(-7) M) or genistein (10(-5) M), which is an inhibitor of protein kinases, in the reaction mixture. The presence of regucalcin (0.1-0.5 microM) caused a significant decrease in protein phosphorylation in the cytosol from normal and regenerating rat livers. The effect of regucalcin (0.5 microM) was completely abolished by the addition of anti-regucalcin monoclonal antibody (50 ng/ml). The elevation of protein phosphorylation in regenerating rat liver was significantly enhanced by the presence of anti-regucalcin monoclonal antibody (50 ng/ml). The effect of regucalcin in decreasing protein phosphorylation in the cytosol of regenerating rat liver was not seen in the presence of the antibody. The present study demonstrates that protein kinase activity, is enhanced in the cytosol of regenerating rat liver, and that endogenous regucalcin has an inhibitory role in the enhancement of protein phosphorylation by various protein kinases.     

Effects of norepinephrine on transmembrane calcium transport in rat mast cells

The effect of norepinephrine on transmembrane passage of calcium in rat peritoneal mast cells, was studied in an in vitro system. It was found the histamine release from mast cells induced by the ionophore A 23 187 in normal calcium medium and compound 48/80 in a calcium-free medium was suppressed by 10(-3) M norepinephrine but not at concentrations in the range 10(-5)-10(-4) M. When the secretory process is totally dependent on the presence of calcium in the incubation medium, i.e. calcium-depleted cells, 10(-5) M norepinephrine suppresses the histamine release induced by low concentrations of compound 48/80. The effect of norepinephrine (10(-5) M) on strontium-induced "spontaneous' histamine release was also studied. It was found that norepinephrine (10(-5) M) totally inhibits the progressive histamine release induced by strontium. It is possible to evoke secretion in a calcium-free medium, and subsequent introduction of Ca2+ will result in optimal histamine release. This demonstrates a secretory process in which we can distinguish between utilization of endogenous versus exogenous calcium. The release that is dependent on extracellular calcium is inhibited by norepinephrine (10(-5) M). These data indicate that the suppressive effect of norepinephrine (concentrations less than 10(-3) M) on histamine release from rat mast cells is due to an interference in transmembrane passage of calcium.